Using antibodies against P2Y and P2X receptors in purinergic signaling research

Abstract

The broad expression pattern of the G protein-coupled P2Y receptors has demonstrated that these receptors are fundamental determinants in many physiological responses, including neuromodulation, vasodilation, inflammation, and cell migration. P2Y receptors couple either Gq or Gi upon activation, thereby activating different signaling pathways. Ionotropic ATP (P2X) receptors bind extracellular nucleotides, a signal which is transduced within the P2X protein complex into a cation channel opening, which usually leads to intracellular calcium concentration elevation. As such, this family of proteins initiates or shapes several cellular processes including synaptic transmission, gene expression, proliferation, migration, and apoptosis. The ever-growing range of applications for antibodies in the last 30 years attests to their major role in medicine and biological research. Antibodies have been used as therapeutic tools in cancer and inflammatory diseases, as diagnostic reagents (flow cytometry, ELISA, and immunohistochemistry, to name a few applications), and in widespread use in biological research, including Western blot, immunoprecipitation, and ELISPOT. In this article, we will showcase several of the advances that scientists around the world have achieved using the line of antibodies developed at Alomone Labs for P2Y and P2X receptors.

Sensory–sympathetic coupling in superior cervical ganglia after myocardial ischemic injury facilitates sympathoexcitatory action via P2X7 receptor

“…P2X 7 receptor is expressed in SCG [20,41]. In some tissue P2X 7 antibody gives positive reaction in P2X 7 KO mice probably interacting with splice variants of the P2X 7 receptors [8]. Our results showed that MI rats displayed higher levels of P2X 7 immunoreactivity, mRNA, and protein in SCG.…”

“…After washed by PBS for three times, the sections were incubated in 3 % H 2 O 2 for 10 min to block the endogenous peroxidase. Following rinses in PBS, sections were incubated with 5 % BSA for 60 min, then the sections were incubated with rabbit anti-P2X 7 (1:100; Alomone Labs, Jerusalem, Israel) [8], rabbit anti-P2X 3 (1:2,500, Millipore International, Inc, USA), mouse anti-GS (1:100 dilution; Abcam Inc., USA), mouse anti-NeuN (1:200 dilution, Millipore International, Inc, USA), rabbit monoclonal anti-CGRP (1:500 dilution, bioss, CO), rabbit monoclonal anti-SP (1:500 dilution, bioss, CO) or mouse anti-TH (1:1,000 dilution; Abcam International, Inc. USA) diluted in PBS for overnight at 4°C. Sections were rinsed and incubated with biotinylated goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (1:100 dilution; Beijing Zhongshan Biotech.…”

“…P2X 7 receptor plays an important role in functions and diseases of the nervous system [13,40,41]. ATP and P2X 7 receptor are involved in the responses of injury [3,8,9,11,18,41]. P2X 7 blockade reduced brain damage after ischemia [3].This study investigated the effects of P2X 7 receptor on the increased sympathoexcitatory action in SCG caused by MI injury and examined if there is a specific connection between SCG and the cervical dorsal root ganglion (DRG) afferents which would exaggerate the sympathoexcitatory reflex due to activation of P2X 7 receptor during MI injury.…”

Lack of specificity shown by P2Y6 receptor antibodies

“…Of the three antibodies we tested, our examination of the literature, indicated that APR-011 from Alomone Labs has been widely used and has been published several times with beautiful images provided in rat retina (Wurm et al, 2009, Brass et al, 2012, Zhang et al, 2012) and duodenum (Mizumori et al, 2009, Brass et al, 2012) and Western blot data provided in cell lines (Lau et al, 2011, Tamaishi et al, 2011). One reason this antibody is so compelling is its ability to discriminate between cell types and produce specific staining patterns within a tissue.…”

Differential expression of ATP-gated P2X receptors in DRG between chronic neuropathic pain and visceralgia rat models

“…Subsequently, the sections were incubated with horseradish peroxidase (HRP)-marked goat anti-rabbit or donkey antigoat IgG in PBS for 1 h and then reacted with color at room temperature, timely terminating the color reaction, hematoxylin counterstain, dehydration, and transparention and observing under the microscope (Olympus, Japan) and taking photograph. The primary antibodies were rabbit anti-P2X1-5, 7 antibodies, at 1:200 proportional dilution (Alomone Labs, Israel) [15], and goat anti-P2X6 antibody, at 1:100 dilution (Santa Cruz, USA). The second antibodies were HRP-conjugated goat anti-rabbit and donkey anti-goat IgG (1:1000, Santa Cruz Inc., USA).…”